Biological material pre-fixation treatment

ABSTRACT

Disclosed is a controlled autolysis method for making biological tissue substantially acellular by exposing the biological material, prior to any fixation thereof, to at least one buffered solution having a pH in the range from about 5.0 to 8.0 and a temperature in the range from about 12° C. to 30° C. for a sufficient period of time to render at least one region of the biological material substantially acellular while substantially preserving the structural integrity and non-cellular structural components of the biological material. Also disclosed is a method of making a bioprosthetic heart valve using biological material that has been treated by controlled autolysis and a method of treating a mammal having a defective heart valve using a bioprosthetic heart valve made, in part, by controlled autolysis.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 08/229,452,filed Apr. 18, 1994, now U.S. Pat. No. 5,595,571.

FIELD OF THE INVENTION

This invention relates to methods for rendering biological materialssubstantially acellular and for methods of treating biological materialsto inhibit their mineralization after implantation into a human oranimal. In one embodiment, this invention relates to a method ofinhibiting post-implantation mineralization of a bioprosthetic heartvalve.

BACKGROUND OF THE INVENTION

Disorders of the cardiac valves cause significant morbidity andmortality. These disorders affect persons of all ages and can resultfrom congenital or degenerative conditions, as well as from the sequelaeof infections. Stenosis and insufficiency of the aortic or mitral valveshave a greater incidence than stenosis and insufficiency of thetricuspid and pulmonary valves.

Treatment of cardiac valvular disorders can require replacement of thedefective valve with a prosthetic valve. There are two types ofprosthetic heart valves. "Mechanical valves", the first type, arecomposed wholly of materials not derived from living organisms.Mechanical valves currently in use have either a ball-valveconstruction, a tilting disc construction or a hinged leafletconstruction.

"Bioprosthetic valves", the second type of prosthetic heart valves, arecomposed in whole or in part of biological material. Bioprostheticvalves generally comprise a supporting stent and a plurality ofleaflets. The leaflets generally comprise biological material, while thestent, if present, generally comprises non-biological material, at leastin part. The biological material of the leaflets, can be of autologoustissues, such as pericardium, fascia lata or cardiac valves.Alternately, this material can be derived from homologous tissue, suchas non-autologous human tissue for human implantation, or can bexenogeneic.

Each type of prosthetic heart valve has advantages and disadvantages.Mechanical heart valves are durable but they carry a significant risk ofthrombus formation with secondary complications. Chronic anticoagulationtherapy decreases the incidence of thrombotic related events to between1% to 4% per patient year. (Criscitiello, M. and Levine, H.:Thromboembolism and Prosthetic Heart Valves. Hospital Practice. Dec. 15,1992:69-96.) Chronic anticoagulation therapy, however, carries with it arisk of hemorrhage similar in incidence to that of the residual risk forthrombotic events. (Barnhart, G. et al.: Degeneration and Calcificationof Bioprosthetic Cardiac Valves. American Journal of Pathology. 1982,106/1:136-139.)

Bioprosthetic valves initially approximate the hemodynamic properties ofthe natural valve. They carry a smaller risk of complications secondaryto thrombus formation than do mechanical valves, Thus, chronicanticoagulation therapy need not be instituted in most patients.Bioprosthetic valves, however, carry a significantly higher risk ofcalcification than mechanical valves.

Calcification of bioprosthetic valves develops more rapidly in children,which have an incidence of calcification of about 40% to 50% at 4 years,than it develops in adults, which have an incidence of calcification ofbetween 5% to 20% at 10 years. (Carpentier, A. et al.: Techniques forPrevention of Calcification of Valvular Bioprostheses. Circulation 70(suppl I). 1984, I-165 to I-168.) Calcification causes thickening,retraction and reduced mobility of the leaflets and can lead tostenosis, insufficiency or both. Hence, calcification is an importantlimitation on the useful life expectancy of the currently usedbioprosthetic valves. Since treatment of a functionally compromisedbioprosthetic heart valve frequently requires replacement with a newvalve, limitations on the useful life expectancy of a bioprostheticheart valve are both a serious medical problem for the patient and afinancial drain on the medical system.

Several methods to decrease or prevent bioprosthetic heart valvemineralization have been described in several patents since the problemwas identified. Generally, the methods involve treating thebioprosthetic valve with various substances prior to implantation. Amongthe substances reported to work are sulfated aliphatic alcohols,phosphate esters, amino diphosphonates, derivatives of carboxylic acidand various surfactants. Nevertheless, none of these methods have provencompletely successful in solving the problem of post-implantationmineralization. Thus, there remains a need for the bioprosthetic heartvalve resistant to post-implantation mineralization.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a controlled autolysis method for making a biological materialsubstantially acellular, wherein the biological material that is to bemade substantially acellular has structural integrity and comprisescells and non-cellular structural components, and wherein the controlledautolysis method comprises the step of exposing the biological material,prior to any fixation thereof, to at least one buffered solution havinga pH in the range from about 5.0 to 8.0 and a temperature in the rangefrom about 12° C. to 30° C. for a sufficient period of time to render atleast one region of the biological material substantially acellularwhile substantially preserving the structural integrity and non-cellularstructural components of the biological material.

In accordance with another aspect of the present invention, there isprovided a bioprosthetic heart valve, comprising at least one leafletthat is adapted for reciprocal motion from an open position to a closedposition upon blood flow through the valve, the at least one leafletbeing formed, at least in part, of biological material that has beensubjected to controlled autolysis, wherein the biological material thatis subjected to controlled autolysis has structural integrity andcomprises cells and non-cellular structural components.

In accordance with another aspect of the present invention, thebioprosthetic heart valve additionally comprises a generally tubularstent having an inflow end and an outflow end, wherein the at least oneleaflet is positioned in relation to the stent such that the reciprocalmotion of the at least one leaflet occurs as blood flows from the inflowend of the stent through the outflow end of the stent.

In accordance with another aspect of the present invention, there isprovided a method of making a bioprosthetic heart valve, comprising thesteps of (a) subjecting biological material to the controlled autolysismethod of the present invention, wherein the biological materialcomprises a heart valve or a fragment of a heart valve, the heart valveor fragment of a heart valve comprising at least one leaflet, (b) fixingthe biological material, and (c) fabricating the bioprosthetic heartvalve from the biological material by the addition of non-biologicalmaterial.

In accordance with another aspect of the present invention, there isprovided a method of treating a mammal having a defective heart valve,comprising the steps of (a) providing a bioprosthetic heart valvecomprising at least one leaflet that is adapted for reciprocal motionfrom an open position to a closed position upon blood flow through thevalve wherein the leaflet is formed, at least in part, of biologicalmaterial that has been subjected to the control led autolysis method ofthe present invention, (b) removing the defective heart valve from themammal, and (c) implanting the bioprosthetic heart valve in the mammal.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one figure executed in color.Copies of this patent with color figures will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 is an outflow end elevational view of bioprosthetic heart valve.

FIG. 2 is an inflow end elevational view of bioprosthetic heart valve.

FIG. 3 is a photomicrograph of a cross-section of a porcine aortic valveleaflet before treatment at 20X (twenty times magnification).

FIG. 4 is a photomicrograph of a cross-section of a porcine aortic valveleaflet before treatment at 100X (one hundred times magnification).

FIG. 5 is a photomicrograph of a cross-section of a porcine aortic valveleaflet after treatment at 20X (twenty times magnification).

FIG. 6 is a photomicrograph of a cross-section of a porcine aortic valveleaflet after treatment at 40X (forty times magnification).

FIG. 7 is a photomicrograph of a cross-section of a porcine aortic valveleaflet 2 (two) weeks after implantation that had not been treated at40X (forty times magnification).

FIG. 8 is a photomicrograph of a cross-section of a porcine aortic valveleaflet 20 (twenty) weeks after implantation that had not been treatedat 40X (forty times magnification).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In one aspect, the present invention relates to an improvedbioprosthetic heart valve having an improved resistance topost-implantation mineralization. In this aspect, the invention involvesthe use of specially treated biological material. The material istreated by exposing the biological material, prior to fixation, to atleast one buffered solution having a preselected pH range and apreselected temperature range for a sufficient period of time to renderat least one region of the biological material substantially acellularwhile substantially preserving the non-cellular structural components ofthe biological material. This treatment involves the process hereinafterreferred to as "controlled autolysis" and described more fully below.

Selected Definitions:

As used herein, "biological material" comprises cells and non-cellularstructural components derived from at least one of a living organism,the dead body of a human or animal, an organ or part of an organartificially maintained outside the living organism, a cell culturederived from a living organism, a corpse or a carcass, and a combinationof the preceding. Biological material can be artificially manipulatedprior to derivation from the above listed sources, such as by theintroduction of special diets or drugs to the organism, or theintroduction of gene sequences by an appropriate vector. Each biologicalmaterial has a "structural integrity", as defined below.

As used herein, the terms "bovine", "porcine", "ovine" "Macropodidae"and "non-human primate" refer respectively to at least one animal of orrelated to a cow or ox, pig or hog, sheep or goat, a kangaroo, andmonkey or ape or gibbon or chimpanzee or lemur. The terms include, butare not limited to, an unborn animal, young animal, male animal, femaleanimal and pregnant animal, whether occurring naturally, throughselective breeding or artificial insemination, a carcass, a fragmentthereof, and a combination of any of the preceding. As used herein,"human" includes an embryo, a fetus, an unborn individual, a corpse, afragment or tissue of any of the foregoing and a combination thereof.

As used herein, a "buffered solution" refers to an aqueous solutionhaving at least one substance which tends to preserve hydrogen ionconcentration or pH.

As used herein, "cell" means the composite of the membrane structuresenveloping protoplasm and distinguishing the enveloped protoplasm fromthe external environment, wherein the membranes are identifiable byvisual inspection with the aid of light microscopy. The cell can beliving or not living. "Cell" also includes remnants or ghosts of thecomposite of the membrane structures indicating the former presence of aliving cell at or near the location of the remnant or ghost.

As used herein, "membrane structures" refer to lipid layers and lipidbilayers, with or without proteins, carbohydrates, glycoproteins,cholesterol or other substances incorporated into the layers, such asare found enveloping protoplasm from a living cell.

As used herein, "non-cellular structural components" comprisessubstances not enveloped by the composite of the membrane structures ofa cell, even if derived from or secreted by cells. The substancesinclude collagen, elastin, laminin, teninsin, actinin and proteoglycans.

As used herein, "fixation" or "fixing" refers to a process of treatingbiological material so as to preserve the material from natural decay,including decay by autolysis. Fixation includes methods such as exposingthe biological material to glutaraldehyde or formaldehyde.

As used herein, "fragment" means any portion or amount less than thewhole, including disjoined or non-contiguous portions.

As used herein, "heart valve" means at least one of the aortic valve,mitral valve, tricuspid valve and pulmonary valves in a human, anequivalent valve in non-human animals, with or without intimatelyrelated tissue, a fragment thereof and a combination thereof.

As used herein, "region", as applied to biological material, refers tothe whole biological material or any fragment of the biological materialhaving macroscopically identified boundaries. For example, a region of aharvested natural heart valve can be the whole valve, at least oneleaflet, the stent, the adjunct myocardium and a combination thereof.

As used herein, "structural integrity" refers to the natural capacity ofbiological material to perform a physical, as opposed to chemical,function in the organism, such as a compression function, a valvularfunction or a support function.

As used herein, "substantially acellular" and "substantial acellularity"interchangeably mean having at least about 70% (seventy percent) fewercells than the natural or living state of the biological material.Therefore, biological material that has been made substantiallyacellular according to the present invention, has had the absolutenumber of cells reduced by at least about 70% from the natural state.

For the purposes of determining substantial acellularity, only cellsnative to the biological material are counted. Blood borne cellsincluding red cells and platelets, as well as cells from other organismsare not counted.

The number of cells present in biological material is determined byusing visual inspection of the material at about 20X (twenty times) to100x (one hundred times) magnification using light microscopy with orwithout stain, or an equivalent technique. Satisfactory stains includestandard stains known to those with skill in the art, as is appropriateto the specific cell type of the biological material being examined.

Conditions for Controlled Autolysis.

Various aspects of the present invention utilize the process hereinreferred to as "controlled autolysis". "Controlled autolysis" meansmanipulating the physical conditions and storage solutions to whichbiological material is exposed to promote the breakdown of certaincomponents of the biological material while substantially preservingother components due to properties of the autolytic enzymes found in thebiological material. The treatment is performed prior to fixation.

Controlled autolysis can be used as a method of treating biologicalmaterial that will be implanted into a human or animal to inhibitmineralization of regions of the biological material that are prone tomineralization after implantation. The treatment appears to inhibitmineralization by digesting cells, components of which attract mineralssuch as calcium after implantation, thereby allowing for the removal ofthe cells from the biological material. Controlled autolysis can also beused to render biological material substantially acellular for purposesother than inhibiting the post-implantation mineralization, such as fordecreasing the immunogenicity or toxicity of the material.

Conditions important to produce controlled autolysis include pH,temperature, amount of solution per unit of biological material andtime. In general, biological tissue to be processed by controlledautolysis is exposed to at least one buffered solution having apreselected pH range and a preselected temperature range for asufficient period of time to complete the process.

The conditions for controlled autolysis are selected to advantageouslypromote activity of the autolytic enzymes which degrade cells Whileinhibiting, not affecting or promoting to a satisfactory minimal extentthe autolytic enzymes that degrade non-cellular structural components.Further, these conditions advantageously maintain bacterial growth onthe biological material to an acceptable level, preferably preventinglog phase bacterial growth prior to completion of the treatment. Bykeeping the absolute bacteria burden on the biological tissue low,potential toxicity from bacterial components is reduced.

Controlled autolysis can be performed using any of a variety of bufferedsolutions known to those with skill in the art. Suitable bufferedsolutions include phosphate buffers, such as sodium phosphate monobasicand dibasic buffers, and phosphate citrate buffer. Any of a variety ofother buffers well known by those of skill in the art can also be used.In one preferred embodiment, the buffered solution is sodium phosphatemonobasic and dibasic buffers in saline solution at a pH of 7.4.

The aqueous portion of the buffered solution is preferably relativelypure. Water processed through a single distillation is satisfactory.Water processed by reverse osmosis filtration is also satisfactory.Double distilled water and water processed by both distillation andfiltration is preferred but not necessary.

In one embodiment of the present invention, biological material isexposed to the buffered solution by immersion. As cells are degradedfrom the material, the degradation components are dispersed orsolubilized in the buffered solution, thereby desirously leaching out ofthe material. It is therefore advantageous to keep the concentration ofthe cellular degradation components low in the buffered solution topromote this process.

Concentration of cellular degradation components can be kept low byregularly replacing the buffered solution. For example, biologicalmaterial or groups of biological material can be positioned incontainers of buffered solution. Racks carrying the biological materialscan then be placed in different containers of fresh buffered solution asneeded to keep the concentration of cellular degradation components inthe buffered solution surrounding the biological material to asatisfactorily low level.

Alternately, biological material or groups of biological material can bepositioned in containers of buffered solution and fresh bufferedsolution circulated in the container on a continuous or intermittentbasis as needed. A combination of these methods, with or withoutequivalent methods, are contemplated within the scope of the presentinvention.

The preselected pH of the buffered solution can vary within a range fromabout 5.0 to about 8.0, more preferably 6.0 to 7.8. In one preferredembodiment, the preselected pH range is from about 7.2 to about 7.6.Using buffered solutions having a pH above about 7.8 to 8.0disadvantageously promotes the activity of certain autolytic enzymesthat degrade non-cellular structural components.

The preselected temperature range of the buffered solution can vary fromabout 12° C. to about 30° C., more preferably from about 15° C. to about27° C. in one preferred embodiment, the preselected temperature range isfrom about 19° C. to about 23° C.

Temperatures significantly below about 12° C. decrease the activity ofthe autolytic enzymes which degrade cells, thereby disadvantageouslyprolonging the treatment time. Indeed, a temperature of about 5° C. canbe used to preserve the biological tissue by suppressing the activity ofthe autolytic enzymes which degrade cells, among other enzymes.Temperatures above about 30° C. disadvantageously promote the autolyticenzymes that degrade non-cellular structural components, promote thedenaturation of proteins present in the non-cellular structuralcomponents and promote the growth of contaminating bacteria into the logphase before completion of the treatment. Temperatures between about 19°C. and 23° C. work particularly well because they allow the autolyticenzymes which degrade cells to function at a satisfactory rate, whilesuppressing bacterial growth to a satisfactory level.

The period of time sufficient to complete the process of controlledautolysis is judged by testing samples of the biological material todetermine when at least one preselected region of the biologicalmaterial has been rendered substantially acellular while substantiallypreserving the structural integrity and non-cellular structuralcomponents of the biological material. In general, the period of timecan range from about a few hours to many days, depending on thepreselected conditions of pH and temperature, type of biologicalmaterial, quantity of biological material, degree of acellularitydesired and other factors. The endpoint of the treatment can bedetermined by comparing the cellularity of treated biological tissue toa control or by counting the absolute number of cells in the preselectedregion and comparing the number to a known value for such a region.

In one preferred embodiment, the period of time is from about 24 hoursto about 140 hours. In one particularly preferred embodiment, the periodof time is from about 65 hours to about 75 hours. However, in certaincircumstances period as short as 12 hours or shorter may be sufficient.Alternatively, other circumstances can require periods as long as 240hours or longer.

A Bioprosthetic Heart Valve.

Referring now to FIGS. 1 and 2 in detail, there is shown an outflow endelevational view and an inflow end elevational view, respectively, of abioprosthetic heart valve 5 of the present invention. The valve has atleast one leaflet that comprises a biological material. The biologicalmaterial has at least one substantially acellular region, which can beproduced in accordance with the controlled autolysis proceduresdescribed above.

The bioprosthetic heart valve 5 can comprise a stent 20, which can beformed from non-biological material in whole or in part. Thenon-biological material used to form the stent can be any of a varietyof biocompatible materials well known to those with skill in the art.

The stent 20 is generally in the shape of a tube having a round or ovalcross-sectional shape and an inner surface and an outer surface. Thestent 20 can also have any of a number of projections such as 50, whichcan be used to attach the leaflets to the stent or to attach the valveto a vessel wall during implantation or for other reasons. The stentprovides support for the at least one leaflet 10 and is adapted topermit blood flow therethrough.

The substantially acellular region of the biological material ispreselected prior to treatment by the method of making biological tissuesubstantially acellular according to one aspect of the presentinvention. This region can be all of the biological material, the atleast one leaflet, the stent, or a fragment or combination of all of theforegoing.

The at least one leaflet 10 has a base 40 which serves as an attachmentpoint to the stent 20 and an edge 30. The at least one leaflet 10 isadapted for reciprocal motion from an open position to a closed positionupon blood flow through the stent. In the closed position, the edge 30serves as a point of coaptation with other leaflet edges, where present,or the stent, to inhibit retrograde flow of blood.

The biological material of the bioprosthetic heart valve can be derivedfrom tissue of any of a variety of animals. Thus, tissues from a bovine,porcine, ovine, Macropodidae, nonhuman primate and human sources or acombination thereof can be used. In one preferred embodiment, thebiological material is porcine in origin.

The biological material can be derived from a variety of tissues andorgans including, but not limited to diaphragm, pericardium, heartvalve, or a fragment or combination of all of the foregoing. In onepreferred embodiment, the biological material is a heart valve, afragment of a heart valve and a combination thereof.

Method of Making a Bioprosthetic Heart Valve.

One of the aspects of the present invention relates to a method ofmaking a bioprosthetic heart valve utilizing controlled autolysis. Themethod of making a bioprosthetic heart valve involves the provision ofbiological material as described above. The biological material hasstructural integrity and comprises both cells and non-cellularstructural components. In one preferred embodiment, the biologicalmaterial is derived from porcine heart valves.

The biological material is obtained from sources well known to those inthe art. In one preferred embodiment, the biological material is derivedfrom porcine heart valves obtained from a slaughterhouse which issubject to inspections specifically for the purpose of insuring theprovision of suitable quality biological material. The biologicalmaterial does not necessarily need to be harvested under sterileconditions, but it is preferred that such material be harvested underclean conditions to reduce the amount of contamination.

After harvesting, the biological material is immediately stored in abuffered solution having a temperature range from about 3°-10° C. Anespecially preferred pH range for this solution is from about 7.0 toabout 7.8. Suitable buffers for storage are well known to those withskill in the art; these include any of a variety of phosphate andnon-phosphate buffers. The temperature and pH of these buffers areselected so as to preserve the fresh state of the biological tissueuntil the biological tissue can be further processed by controlledautolysis. Thus, for certain buffers and conditions, the pH andtemperature can be outside of these preferred ranges.

The biological tissue can be stored for an extended period of time, suchas from days to weeks. However, most preferably, the biological tissueis further processed within hours of being harvested for maximumpreservation of the structural components of the tissue. In aparticularly preferred embodiment, the biological material begins thenext step, a rinsing step, within 2-10 hours after harvesting.

When the biological tissue is ready for processing by controlledautolysis, it is first rinsed by placement in fresh buffered solutionfor several hours to a few days in order to reduce the amount ofcontaminants present on the biological material. In one preferredembodiment, the biological tissue is rinsed for approximately 24 hours.

The rinsing solution has the same temperature and pH ranges as thestorage solution, such that the fresh state is still maintained whilereducing the amount of contaminants. In a preferred embodiment, thetemperature of the solution is about 3°-7° C. and the pH is from about7.2-7.6. Buffer solutions suitable for rinsing are the same as thosesuitable for storing the freshly harvested biological material, and arewell known to those with skill in the art.

The amount of rinsing solution varies with the amount of biologicaltissue to be rinsed. When the biological material is porcine heartvalves, approximately 100-300 ml of rinsing solution is adequate. In onepreferred embodiment where the biological material is porcine heartvalves, approximately 100 ml of rinsing solution per valve is used.

Controlled autolysis is initiated by transferring the biologicalmaterial to a container of fresh buffered solution or replacing therinsing solution with fresh buffered solution. This constitutes thefirst treatment stage of controlled autolysis. The amount of bufferedsolution again varies with the amount of biological material but shouldbe enough to cover the material completely and allow for dilution of theextracted cells during the process. When the biological material isporcine heart valves, approximately 100-300 ml of buffered solution isadequate. In one preferred embodiment where the biological material isporcine heart valves, approximately 200 ml of solution per valve isused. Thus, where approximately 40 porcine heart valves are to beprocessed by controlled autolysis in one container, approximately 8000ml total of buffered solution will be present in the container.

The buffered solution can be an aqueous solution of any of a variety ofbuffers. Suitable buffered solutions include phosphate buffers, such assodium phosphate monobasic and dibasic buffers, and phosphate citratebuffer. Any of a variety of other buffers well known by those of skillin the art can also be used. In one preferred embodiment, the bufferedsolution is sodium phosphate monobasic and dibasic buffers in salinesolution.

The buffered solution should maintain the biological tissue at a pH ofbetween 6.0 and 8.0. In a preferred embodiment the pH is between 7.0 and7.8. When the biological tissue is porcine heart valves, it is preferredthat the pH be between 7.2 and 7.6.

The temperature of the buffered solution can vary from about 12° C. toabout 30° C., more preferably from about 15° C. to about 27° C. In onepreferred embodiment, the temperature range of the buffered solution isfrom about 19° C. to about 23° C.

Temperatures significantly below about 12° C. decrease the activity ofthe autolytic enzymes which degrade cells. Temperatures above about 30°C. disadvantageously promote the autolytic enzymes that degradenon-cellular structural components, promote the denaturation of proteinspresent in the non-cellular structural components and promote the growthof contaminating bacteria into the log phase before completion of thetreatment. Temperatures between about 19° C. and 23° C. workparticularly well because they allow the autolytic enzymes which degradecells to function at a satisfactory rate, while suppressing bacterialgrowth to a satisfactory level.

Where the biological tissue is porcine heart valves, the valves arearranged in the container in a single layer and covered by the bufferedsolution. Gentle agitation is used to promote removal of the cells fromthe valves.

The biological tissue is maintained in the container for a period oftime ranging from hours to days. In a preferred embodiment, thebiological material is maintained in the container for approximately 24hours.

Next, the valves are rinsed in fresh buffered saline having the same oran equivalent buffer as is used in the first treatment stage ofcontrolled autolysis and transferred to another container havingbuffered solution with parameters similar to the first treatment stageof controlled autolysis. Essentially, the first stage of controlledautolysis is then repeated, constituting a second stage of controlledautolysis.

After completion of the second stage of controlled autolysis, additionalstages may be performed if necessary to achieve the desired level ofacellularity. In a preferred embodiment, three stages are performedtotaling a period of approximately 72 hours total for treatment bycontrolled autolysis.

After completion of controlled autolysis, or during the process, asample or samples of the biological material are checked for the levelof acellularity by methods well known to those with skill in the art andvarying by the type of biological tissue being treated. For example,microscopic examination using cryosectioning or paraffin sectioningtechniques or other techniques may be used.

Once the treatment is completed, the biological material is processedand fabricated into finished bioprosthetic heart valves using standardtechniques well known to those with skill in the art. This may includefixation, such as by glutaraldehyde, sterilization with radiation orchemicals, and the addition of non-biological material to the biologicalmaterial, such as by the addition of a stent, all processes well knownto those with skill in the art. An example of a finished bioprostheticvalve made according to this method is found in the section titled "ABioprosthetic Heart Valve", above.

Method of treating a human or animal with a defective heart valve.

In another aspect, the present invention is a method of treating amammal with a defective heart valve. The method of treating involves theprovision of a heart valve made according to the method of described inthe section titled "Method of Making a Bioprosthetic Heart Valve." Thedefective heart valve is removed and the bioprosthetic heart valveimplanted in the mammal.

Method of Treating Biological Material For Incorporation Into aBioprosthetic Heart Valve.

In another aspect, the present invention is a method of utilizingcontrolled autolysis to treat biological material that is incorporatedinto a bioprosthetic heart valve, or that will be incorporated into abioprosthetic heart valve, to inhibit post-implantation mineralizationof the bioprosthetic heart valve. The method of treating the biologicalmaterial involves providing biological material, as described above. Thebiological material has structural integrity and comprises both cellsand non-cellular structural components. The biological material isexposed, prior to fixation, to at least one buffered solution having apreselected pH range and a preselected temperature range for asufficient period of time to render at least one preselected region ofthe biological material substantially acellular while substantiallypreserving the structural integrity and non-cellular structuralcomponents of the biological material as is described more fully above.In addition to these steps, the biological material can be furtherprocessed or altered, as also described in the sections above.

Method of Making Biological Material Substantially Acellular.

In a further aspect, the present invention is a method of utilizingcontrolled autolysis to make biological material substantiallyacellular. The method involves providing biological material. Thebiological material has structural integrity and comprises both cellsand non-cellular structural components. The biological material isexposed, prior to fixation, to at least one buffered solution having apreselected pH range and a preselected temperature range for asufficient period of time to render at least one preselected region ofthe biological material substantially acellular while substantiallypreserving the structural integrity and non-cellular structuralcomponents of the biological material.

The biological material suitable for treatment by this method can bederived from any of a variety of animal tissue. Thus, tissues from abovine, porcine, ovine, Macropodidae, nonhuman primate and human sourcesor a combination thereof can be used. In one preferred embodiment, thebiological material is porcine in origin.

The biological material can be derived from a variety of tissues andorgans including, but not limited to diaphragm, pericardium, heartvalve, umbilical cord, artery, vein, facia lata, dura mater, tendon,ligament, tympanic membrane, or a fragment or combination of all of theforegoing. In one preferred embodiment, the biological material is aheart valve, a fragment of a heart valve and a combination of theforegoing.

Referring now to FIG. 3, there is provided a photomicrograph of across-section of a porcine aortic valve leaflet, which is biologicalmaterial suitable for use in the method of making a bioprosthetic heartvalve in accordance with one aspect of the present invention. Thebiological material was stained with hematoxylin and eosin usingstandard techniques and the photomicrograph taken under low power, 20X(twenty times magnification). The cell nuclei present in the tissue arerepresented by the small dark purple spots visible throughout most areasof the tissue.

Referring now to FIG. 4, there is provided a photomicrograph of across-section of a porcine aortic valve leaflet, similar to the porcineaortic valve leaflet shown in FIG. 3, but taken under 100X (one hundredtimes magnification). The cell nuclei present in the biological tissueare again represented by the small dark purple spots visible throughoutmost areas of the tissue. The tissue shown in both FIG. 3 and FIG. 4 hadnot been subject to the process of controlled autolysis as describedherein.

Referring now to FIGS. 5 and 6, there are provided photomicrographs of across-section of a porcine aortic valve leaflet, similar to FIGS. 3 and4, but taken after the biological tissue has been subject to the processof controlled autolysis as described herein in the method of makingbiological tissue acellular. FIG. 5 was taken under low power, 20X(twenty times magnification) and FIG. 6 was taken under 40X (forty timesmagnification). The biological tissue was stained by standard techniquessuch that collagen appears pink to red and elastin appears black.

Note that in both FIGS. 5 and 6, there appears to be a near completeabsence of cells nuclei indicating that the tissue has been renderedsubstantially acellular by the controlled autolysis treatment. Further,note that the non-cellular structural components of the biologicalmaterial, here collagen and elastin, appear to have been substantiallypreserved and that the structural architecture of the leaflet issubstantially intact.

Method of Treating Biological Material To Inhibit Post-ImplantationMineralization.

In one aspect, the present invention is a method of utilizing controlledautolysis to treat biological material that will be implanted into ahuman or animal to inhibit mineralization of regions of the biologicalmaterial that are prone to mineralization after implantation into thehuman or animal. The method comprises providing biological material asdescribed above and treating the biological material as described in thesection titled "Method of Making Biological Material SubstantiallyAcellular".

Referring now to FIG. 7, there is provided a photomicrograph of across-section of a porcine aortic valve leaflet, taken under 40X (fortytimes magnification). The biological tissue did not undergo treatment bycontrolled autolysis, as described herein, but instead was processed bystandard techniques. The leaflet was placed in a subcutaneous pocket inthe dorsum of a juvenile rat for two weeks. Upon removal, it wassectioned and stained by the method of Von Kossa, which visualizesmineralized material by depositing reduced silver in association withphosphate salts, in this case presumably calcium phosphate. The tissuewas counterstained with hematoxylin and eosin. Note that Von Kossapositive material is present throughout the biological material,indicating considerable mineralization of the biological material.

Referring now to FIG. 8, there is provided a photomicrograph of across-section of a porcine aortic valve leaflet, taken under 40X (fortytimes magnification). The biological tissue Was processed by controlledautolysis, as described herein. The leaflet was used in the fabricationof a complete valve and implanted in a sheep in the mitral position fora period of 20 (twenty) weeks. Upon removal, it was sectioned andstained by the method of Von Kossa and counterstained with hematoxylinand eosin.

The dark purple spots in FIG. 8 represent cell nuclei of host cells thathave attached to the surface of the leaflet and that have migrated intothe leaflet. The small black spots represent normal background stainingassociated with the Von Kossa technique. Note that this section shows noevidence of intrinsic leaflet mineralization.

Example: Making a Bioprosthetic Heart Valve

A bioprosthetic heart valve was made according to the method describedabove. Forty fresh porcine aortic valves were obtained from theslaughterhouse. The valves were harvested under clean, but not sterile,conditions using techniques well known in the art. The valves wereimmediately stored in a solution of phosphate buffered saline at atemperature of 5°-10° C. to preserve the fresh state.

Approximately 4 hours after harvesting, the valves were rinsed byplacing them in a phosphate buffered saline solution of approximately100 ml per valve and having a pH of approximately 7.4 for approximately24 hours at 3°-7° C. to reduce the amount of contaminants present on thevalves.

The valves were then transferred to a container having approximately 200ml of phosphate buffered saline solution per valve for a total ofapproximately 8000 ml, at a pH of 7.4 and a temperature of 21°±2° C. Thevalves were arranged in the container in a single layer and covered bythe buffered solution. Gentle agitation was initiated to promote removalof the cells from the valves. The valves were maintained in thecontainer for approximately 24 hours.

Next, the valves were rinsed in fresh buffered saline and transferred toanother container having approximately 200 ml of fresh phosphatebuffered saline per valve, for a total of approximately 8000 ml, at a pHof 7.4 and a temperature of 21°±2° C. Once again, gentle agitation wasused and the valves remained in the container for approximately 24hours.

Next, the valves were again rinsed in fresh buffered saline andtransferred to another container having approximately 200 ml of freshphosphate buffered saline per valve, for a total of approximately 8000ml, at a pH of 7.4 and a temperature of 21°±2° C. As before, gentleagitation was used.

After an additional 24 hours, a random sample of four valves wasselected for microscopic examination using paraffin sectioningtechniques well known to those with skill in the art. Regions of theleaflets were examined and found to contain less than 2 (two) cells per400X (four hundred times) field. This number of cells is indicative of agreater than 90% loss of cells for porcine heart valves. The treatmentwas, therefore, considered complete and the valves were then fixed,processed and fabricated into finished bioprosthetic heart valves usingstandard techniques well known to those with skill in the art.

After processing and final fabrication, the bioprosthetic heart valveswere tested by an extended accelerated fatigue test, according tostandard techniques for the industry. Adequate performance at a level of200 million cycles indicates that the valve tested retains the necessarystructural integrity. The valves made according to the present inventionwere found to perform adequately for greater than 200 million cycles.Using this technique, greater than 75% of the batches are madesatisfactorily acellular by approximately 72 hours, while maintainingnecessary structural integrity.

Although this invention has been described in terms of certain preferredembodiments, other embodiments that are apparent to those of ordinaryskill of art are also within the scope of the invention. Accordingly,the scope of the invention is intended to be defined only by referenceto the appended claims.

We claim:
 1. A bioprosthetic heart valve, comprising:at least oneleaflet that is adapted for reciprocal motion from an open position to aclosed position upon blood flow through the valve; the at least oneleaflet being formed, at least in part, of biological material that hasbeen subjected to controlled autolysis, wherein the biological materialthat is subjected to controlled autolysis has structural integrity andcomprises cells and non-cellular structural components, and wherein thecontrolled autolysis comprises exposing the biological material, priorto any fixation thereof, to at least one buffered solution having a pHin the range from about 5.0 to 8.0 and a temperature in the range fromabout 12° C. to 30° C. for a sufficient period of time to facilitatedegradation of cells by autolytic enzymes within the cells so as torender at least one region of the biological material substantiallyacellular while substantially preserving the structural integrity andnon-cellular structural components of the biological material.
 2. Thebioprosthetic heart valve of claim 1, wherein the bioprosthetic valveadditionally comprises a generally tubular stent having an inflow endand an outflow end, and wherein the at least one leaflet is positionedin relation to the stent such that the reciprocal motion of the at leastone leaflet occurs as blood flows from the inflow end of the stentthrough the outflow end of the stent.
 3. The bioprosthetic heart valveof claim 2, wherein the stent has a generally circular cross-section. 4.The bioprosthetic heart valve of claim 2, wherein the stent has an innersurface and an outer surface and wherein the at least one leaflet isattached to the inner surface.
 5. The bioprosthetic heart valve of claim2, wherein the stent comprises non-biological material.
 6. Thebioprosthetic heart valve of claim 1, wherein the at least one region ofthe biological material that is rendered substantially acellular issubstantially all of the leaflet.
 7. The bioprosthetic heart valve ofclaim 1, wherein the biological material is porcine.
 8. Thebioprosthetic heart valve of claim 7, wherein the biological material isa leaflet of a heart valve or a fragment of a leaflet of a heart valve.9. The bioprosthetic heart valve of claim 1, wherein the biologicalmaterial is a heart valve or a fragment of a heart valve.
 10. Thebioprosthetic heart valve of claim 1, wherein the controlled autolysisadditionally comprises the step of fixing the biological material afterthe exposing step.
 11. The bioprosthetic heart valve of claim 1, whereinthe biological material is derived from at least one animal selectedfrom the group consisting of a bovine, a porcine, an ovine, aMacropodidae, a non-human primate, a human and a combination of any ofthe foregoing.
 12. The bioprosthetic heart valve of claim 1, wherein thebiological material is at least one material selected from the groupconsisting of diaphragm, pericardium, heart valve, umbilical cord,artery, vein, fascia lata, dura mater, tendon, ligament, tympanicmembrane, a fragment of any of the foregoing, and a combination of anyof the foregoing.
 13. The bioprosthetic heart valve of claim 1, whereinthe non-cellular structural components include at least one structuralcomponent selected from the group consisting of collagen, elastin,laminin, teninsin, actinin, proteoglycans, a fragment of any of theforegoing, and a combination of any of the foregoing.
 14. Abioprosthetic heart valve, comprising:at least one leaflet that isadapted for reciprocal motion from an open position to a closed positionupon blood flow through the valve; the at least one leaflet beingformed, at least in part, of biological material that has been subjectedto controlled autolysis, wherein the biological material that issubjected to controlled autolysis has structural integrity and comprisescells and non-cellular structural components, and wherein the controlledautolysis consists essentially of exposing the biological material,prior to any fixation thereof, to at least one solution consistingessentially of a buffer having a pH in the range from about 5.0 to 8.0and a temperature in the range from about 12° C. to 30° C. for asufficient period of time to facilitate degradation of cells byautolytic enzymes within the cells so as to render at least one regionof the biological material substantially acellular while substantiallypreserving the structural integrity and non-cellular structuralcomponents of the biological material.